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Nucleophosmin binding on the mouse X chromosomes

ABSTRACT: In mammals, one of the female X chromosomes and all imprinted genes are expressed exclusively from a single allele in somatic cells. To evaluate structural changes associated with allelic silencing, we have applied a recently developed Hi-C assay that uses DNase I for chromatin fragmentation to mouse F1 hybrid systems. Results We find radically different conformations for the two female mouse X chromosomes. The inactive X has two superdomains of frequent intrachromosomal contacts separated by a boundary/hinge region. Comparison with the recently reported bipartite 3D structure of the human inactive X shows that the genomic content of the superdomains differs between species, but part of the hinge is conserved and located near the Dxz4/DXZ4 locus. In mouse, the hinge region also contains a minisatellite Ds-TR, and both Dxz4 and Ds-TR appear to be anchored to the nucleolus. Genes that escape X inactivation do not cluster but are located near the periphery of the 3D structure, as are regions enriched in CTCF or RNA polymerase. Fewer short-range intrachromosomal contacts are detected for the inactive alleles of genes subject to X inactivation, compared to the active alleles and to genes that escape X inactivation. This pattern is also evident for imprinted genes, in which more chromatin contacts are detected for the expressed allele. Conclusions By applying a novel Hi-C method to map allelic chromatin contacts, we discover a specific bipartite organization of the mouse inactive X chromosome that probably plays an important role in maintenance of gene silencing.

ORGANISM(S): Mus musculus

PROVIDER: GSE71903 | GEO | 2015/08/11

SECONDARY ACCESSION(S): PRJNA292428

REPOSITORIES: GEO

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Project description:A subset of genomic regions, including one of the female X chromosomes and all imprinted genes, are expressed exclusively from a single allele in somatic cells of mammals. To evaluate structural changes associated with allelic silencing, we used mouse F1 hybrid systems in which X inactivation is skewed and alleles can be distinguished based on single nucleotide polymorphisms to analyze chromatin contacts by a new Hi-C assay that uses DNase I for chromatin fragmentation. Both DNase Hi-C and in situ DNase Hi-C reveal a radically different conformation between the two female mouse X chromosomes. Two superdomains of frequent intrachromosomal contacts separated by a hinge region are specifically observed on the inactive X chromosome both in vivo (brain) and in vitro (Patski cells). A bipartite 3D organization of the inactive X has been also reported in human lymphoblastoid cells, suggesting that this conserved structure probably plays an important role in maintenance of gene silencing on the inactive X. Interestingly, we found that the genomic content of the superdomains differs between species, but that part of the hinge region is conserved and located near the Dxz4/DXZ4 locus that binds CTCF on the inactive X. In mouse, the hinge region also contains a minisatellite Ds-TR adjacent to a promoter with strong CTCF binding. Both Dxz4 and Ds-TR bind nucleophosmin and are enriched in nucleolus-associated chromatin, suggesting anchoring to the nucleolus. Genes that escape X inactivation do not cluster but are preferentially located near the periphery of the 3D structure. Regions enriched in CTCF or RNA polymerase are also preferentially located on the periphery of the inactive X while LINE1 elements are preferentially on the interior. Genes subject to silencing exhibit fewer detectable intrachromosomal contacts than escape genes. This transcription-coupled pattern is also evident for imprinted genes, in which more chromatin contacts are detected on the expressed allele, suggesting greater constraint on the organization of expressed genomic regions.

Project description:During interphase, the inactive X chromosome (Xi) adopts an unusual 3D configuration known as the Barr body and is largely transcriptionally silent. Despite the importance of X inactivation, little is known about the 3D configuration of Xi and its relationship to gene silencing. We recently showed that in humans, Xi exhibits two distinctive structural features. First, Xi is partitioned into two huge intervals, called superdomains, such that pairs of loci in each superdomain show an enhanced contact frequency with one another. The boundary between the two superdomains lies near DXZ4, a macrosatellite repeat spanning ~300kb, whose Xi allele extensively binds the protein CTCF. Second, Xi exhibits extremely large loops, up to 77Mb long, called superloops. DXZ4 lies at the anchor of several superloops. Here, we use 3D mapping to study the structure of Xi, focusing on the role of DXZ4. We show that superloops and superdomains are conserved across mammals. We develop a novel variant of our in situ Hi-C protocol, dubbed COLA (COncatemer Ligation Assay) to probe the higher order structures formed by the superloops. In COLA, in situ proximity ligation of multiple extremely short fragments produced by the enzyme CviJI is used to efficiently map simultaneous proximity among three or more loci. Using data from Hi-C and COLA, we demonstrate that DXZ4 and other superloop anchors tend to co-locate simultaneously within the same cells, a result that is confirmed by 3D-FISH. Finally, we examine the effects of deleting DXZ4 from Xi in human cells. Using in situ Hi-C, microscopy, and RNA-FISH, we show that superdomains and superloops disappear; that Xi frequently dissociates into multiple separate structures; and that transcriptional silencing on Xi is compromised. Deletion of DXZ4 from the active X chromosome (Xa) has no such effect. Thus, DXZ4 is essential for proper folding and silencing of Xi. Hi-C protocol was used on wildtype and DXZ4-deleted cells to examine the structure of Xi. A novel variant of our in situ Hi-C protocol, dubbed COLA (COncatemer Ligation Assay), was developed to probe the higher order structures formed by the superloops. This series also includes RNA-seq data on Retinal Pigmented Epithelial Cells (hTERT-RPE1). At the time of submission, processed data were available only for the RNA-seq samples. Submitter states that processed data files for HiC samples will be added to this series in the future.

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Nucleophosmin binding on the mouse X chromosomes

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